重组α-乙酰乳酸脱羧酶的表达及部分酶学特性  

Expression of Recombinant αAcetolactate Decarboxylase and Its Characterization

作　　者：尹东[1] 卢大宁[1] 黄百渠[1] 李彦舫[2] 

机构地区：[1]东北师范大学遗传与细胞研究所 [2]中国人民解放军农牧大学

出　　处：《生物化学与生物物理学报》1998年第4期325-330,共6页

摘　　要：用聚合酶链式反应（ＰＣＲ）方法以短芽胞杆菌（Ｂａｃｉｌｕｓｂｒｅｖｉｓ）基因组ＤＮＡ为模板，克隆出０．９７ｋｂ的ＤＮＡ片段，经ＤＮＡ测序证明是α－乙酰乳酸脱羧酶（α－ＡＬＤＣ）基因。将该基因重组到质粒ｐＢＶ２２０中，构建了重组表达质粒ｐＢＶＹＩ，转化大肠杆菌ＤＨ５α后，经筛选得到能表达α－ＡＬＤＣ活性的转化子细胞。酶活检测表明重组的α－ＡＬＤＣ的活性是供体菌的１００００倍。从转化子细胞的抽提液中纯化了重组α－ＡＬＤＣ后，研究了其部分酶学特性。α－ＡＬＤＣ活性可被Ｍｎ２＋、Ｓｎ２＋增强，被Ｚｎ２＋、Ｃｄ２＋、Ｆｅ２＋、Ｃｏ２＋、Ｃｕ２＋抑制。氨基酸修饰剂对α－ＡＬＤＣ活性有不同程度的抑制作用。其最适ｐＨ值为５．５。αAcetolactate decarboxylase(αALDC)gene has been cloned from Baillus brevis using PCR amplification. The amplified 0.97 kb DNA fragment was confirmed to be known αALDC gene by DNA sequencing. The fragment was inserted into the vector pBV220 to construct an expression plasmid pBVYI. This recombinant plasmid over expressed αALDC in E.coli DH5α. The αALDC activity of recombinant bacterium was 10 000fold higher than that of Bacillus brevis. After purification, the properties of the recombinant αALDC were studied. The activity of this enzyme could be stimulated by Mn2+, Sn2+ and inhibited by Zn2+, Cd2+, Fe2+, Co2+, Cu2+. Moreover, amino acid modifiers could inhibit differently its activity. The optimum pH of the enzyme reaction was 5.5.

关 键 词：乙酰乳酸脱羧酶 基因克隆 表达 酶学特性 

分 类 号：Q557[生物学—生物化学] Q78